Capacity adaptation of Hediste (Nereis) diversicolor embryogenesis to salinity, temperature and copper

Marine Environmental Research 29 (1990) 227-243

Capacity Adaptation of Hediste (Nereis) diversicolor Embryogenesis to Salinity, Temperature and Copper P. T. E. O z o h * & N. V. J o n e s Institute of Estuarine and Coastal Studies, School of Biological Sciences, University of Hull, HU6 7RX, UK (Received 4 November 1988; revised version received 26 October 1989; accepted 10 November 1989) ABSTRACT Fertilization in Hediste diversicolor was capacity adapted to high salinity, low to high temperatures (0 to 28"2°C), and to low doses of copper (5 to 75 ltg litre - t ) but not to low salinity below 7.63%o and copper doses of 100 ltg litre- x. The 97.7% fertilization success occurred from 16"6 to 18"4°C and from 19"6 to 24.1%o salinity. The cleavage stage was capacity adapted to only low temperature, intermediate salinity, and high temperature and high salinity inhibited it. The 82.95% cleavage success occurred at 10"2°C and 18"92%~ salinity. Cleavage was the most sensitive stage in the embryogenesis and added copper of 5 to 751aglitre -1 inhibited it. The larvae that hatched out under copper treatments were non-motile and their cilia beat with much reduced rhythmicity.

INTRODUCTION Quantifying the effects o f environmental stressors o f salinity, temperature and pollution on the fertilization and embryogenesis of Hediste (Nereis) diversicolor has not been carried out despite the immense values of such studies. The polychaetes are cosmopolitan in distribution and they are one o f the important species in the marine and estuarine environments. Their use as bioassay organisms is a logical one (Reish, 1977). Their physiology and ecological diversity provide a basis for obtaining a greater variety of toxicity *Present address: Biology Programme, School of Science, Abubakar Tafawa Balewa University, PMB 0248, Bauchi, Nigeria. 227 Marine Environ. Res. 0141-1136/90/$03"50 © 1990 Elsevier SciencePublishers Ltd, England. Printed in Great Britain

228

P. T. E. Ozoh, N. V. Jones

information since the environmental requirements and biological factors influence the responses (Maciorowski & Clarke, 1977). Capacity adaptation which involves the change in magnitude of a biological response resulting from a change in the shape (capacity) of a response surface such that the absolute magnitude of a maximum response either increases or decreases is an ideal tool for such studies (Alderdice, 1972). Hediste diversicolor adult is an integrator of heavy metals in estuaries and also an indicator species which occurs in the least saline parts of estuaries where other potential indicator species are excluded (Smith, 1956; Bryan et al. 1985). The purpose of this study is to explore the potential of this worm as 'a sentinel organism' useful for the monitoring of water quality in estuaries.

MATERIALS A N D METHODS Hediste diversicolor exists as one of the dominant species in estuaries. A

review of its distribution in Europe is given in Table 1. The data shown in Table 2 indicate the effects of salinity on its developmental stages collected from both field and laboratory studies.

Worm husbandry The sediments containing mature worms were collected from Grimsby, U K (Grid Ref. 288 109 T.F., O.S.) between March and April (a peak period for sperm and ova maturation). Mature Hediste diversicolor were identified by size and green coloration, and hand picked to remove the attached sediment with jets of water in the laboratory. The clean worms were kept in 15.25%o TABLE 1 Hediste (Nereis) diversicolor Zoogeographical Salinities in Major European Estuaries Estuaries

Scandinavia (Baltic Sea) Bay of Kilonia Southern Coast of Finland and Isefjord, Denmark English River Tamar, Calstock Humber, United Kingdom Alexandra Dock (Hull) Alexandra Dock (Hull) Grimsby

Salinity (%0)

Source

Schlieper (1929) 4 4

Segestrale (1957), Smith (1955a)

0-5 to 30 8"5 to 34 5 to 15 5 to 17 19 to 26

Smith (1955b) Gameson (1982) Fernandez (1983) Ozoh (1986) Ozoh (1986)

Capacity adaptation of H. diversicolor to salinity, temperature, copper

229

TABLE 2 Ranges in Salinities and Developmental Stages of Hediste (Nereis) diversicolor

Location/ Estuaries Scandinavia Scandinavia Scandinavia Scandinavia Scandinavia Humber, UK

Ranges of salinity (%) Critical salinity Below 3.4

State of development

Ripening of gametes H. diversicolor cannot reproduce Wide ranging, 8-27 Fertilization 8-27 Cleavage 8-33-5 Larvae 7.5-26 Adult

Source

Smith (1964) Begucki (1954, 1963) Smith (1964) Smith (1964) Smith (1964) Ozoh (1986)

salinity at 12°C, aerated in glass containers to condition them for 24 h before use. The coelomic fluid of the worm is iso-osmotic with 10%o salinity (Oglesby, 1970). In Grimsby where the worms were collected, salinity ranged from 19 to 26%0 (Table 1). A balance was struck between the iso-osmotic salinity of the coelomic fluid and the prevailing salinity at Grimsby by conditioning them to 15"25%o. In the Humber, temperatures ranged from 2 to 20°C (Gameson, 1982).

Bioassay technique No baseline data exist on the effect of salinity on Humber worms. A pilot study was started by dilution of 30.5%0 salinity sea water with glass distilled tapwater. The following 3.86, 7.63, 9.15, 12-20, 15.25, 18.30, 22.88, 24.40 and 30-5%o salinities were prepared. These salinities were checked with a Beckman salinometer for accuracy. Five female and three male worms were sacrificed by opening them up with scissors to release eggs and sperm in 50 ml distilled water. Each mature female was capable of holding over 50 000 eggs in its coelomic cavity. The released eggs and sperm were stirred with a glass rod and filtered through 500 #M steel mesh to remove debris. The eggs and sperm were pipetted into the prepared salinities and incubated for 24h and aerated at 12°C. About 1000 eggs were in each incubation salinity. After 24h, fertilization was scored by one of the following indices: (1) (2) (3) (4) (5)

Appearance of fertilization membrane. Sperm circulating round an egg. Shedding of zona radiata (egg membrane). Transparency of the egg. Throwing off a jelly layer round an egg. This did not occur often.

P. T. E. Ozoh, N. V. Jones

230

To reduce protozoan and bacterial infestation, one adult worm was added into each incubation dish to feed on these pests. Fertilization was screened with × 5 binocular microscope. Fertilization occurred in salinities 7.63 to 30-5%0. The cleavage screened after 48 h occurred best from 15.25 to 18"3%o salinity (Table 3). It was difficult to screen for fertilization at 3.86%0 because the eggs took in water due to osmotic differences. The salinity dilutions used for the main experiments were 14.6, 21.9 and 29.2%0 prepared by dilution of 29-2%o salinity. These experimental salinities (50, 75 and 100%, respectively) increase orthogonally (Snedecor & Cochran, 1967). These salinity ranges include those occurring where the worms were collected (Table 1) and the experimental temperatures were 12, 17 and 22°C. The Cu concentrations used were 0, 25, 50, 75 and 100 pg litre- x prepared from Cu (NO3) 2 supplied by Fisons Scientific Apparatus, Loughborough, Leics. UK. The standard was l m g m 1 - 1 (1000ppm). The experimental designs were 3 x 3 × 5 combinations (Temperature × salinity x copper) for the determination of fertilization, cleavage and hatching dependence effects on salinity, temperature, copper and their combinations. About seven to eight female and three to five male worms were sacrificed and their eggs and sperm exposed to each salinity regime. All the experiments were replicated four times and 205 eggs were sampled after 24 h for fertilization, 48 h for cleavage and 6 days after the initial day of mixing eggs and sperm for hatching. The hatching was scored by the appearance of a young larva (monotrochophore) or by larval motility. There were 820 eggs screened altogether for each of the developmental stages. The 24 h static renewal of salinity-copper medium was practised to ensure the removal of metabolites and cleaning of incubation glass dishes. Each incubation glass capacity was about 200 ml. TABLE 3 Pilot Study to Determine Fertilization and Cleavage Success in Hediste (Nereis) diversicolor at Different Salinities at 12°C

Salinity (%0)

Fertilization success (%)

Cleavage success (%)

3"86 7'63 9'15 12-2 18"25 18"3 22-88 24-4 30-5

0 18 24 27 54 38 97 100 100

0 4 2 2 60 78 1 1 1

Capacity adaptation of H. diversicolor to salinity, temperature, copper

231

In the Humber, soluble copper occurs in the range of 6 to 8 #g litre-1 (Gameson, 1982). Five and 10 #g litre-1 copper were also prepared in the experimental salinities and these doses were orthogonal to the main experimental designs and may demonstrate the sensitivity of fertilization, cleavage and hatching to copper at levels similar to those encountered in the natural environment.

Statistical analysis The means and standard deviations of fertilization, cleavage and hatching were calculated and subjected to ANOVA. The response-surface contour analysis was used following the method in Davies (1967), and elaborated on in Box (1956), and Box & Wilson (1951) to form a second order polynomial equation of the form; Y = b o x o + blX 1 + b11x I + bx22 X2 -~- b12x1x 2

Y = estimated % of developmental success in fertilization, cleavage or hatching x 1 = temperature factor x 2 = salinity factor bo, bl, 32, bl 1, b12 are coefficients The polynomial equations were converted to canonical equations Y - Y~= B 11 X2 + B22X~2 t o get the maximum yield. To determine capacity adaptation, the canonical equation was used at a fixed percentage yield. For example, 90% fertilization success: Y - 90%

= - B t i X 2 -- B22 X2

The determination of capacity adaptation of developmental stages will show how H e d i s t e d i v e r s i c o l o r is functionally capable to respond to the salinity-temperature stressors c o m m o n in estuaries and how copper concentrations affect them. This will hopefully integrate the laboratory data with realistic field exposures (Tables 1 and 2).

RESULTS

Effect of salinity, temperature and copper on fertilization Below 7.6%o salinity fertilization was not favoured (Table 3). At 3-86%o the eggs took in water osmotically and it was difficult to screen for fertilization

L~ tQ

TABLE 4

Hediste (Nereis) diversicolor F e r t i l i z a t i o n u n d e r Salinity, T e m p e r a t u r e a n d C o p p e r R e g i m e s Temp. (°C)

Copper concentration (l~glitre-1) 0 Salinity (%o)

25

50

.~ .~

14"6

21.9

29"2

14.6

21"9

29.2

14"6

21"9

29.2

99 + 0-87 98+1-0 92 ___+4-95

96 __+ 1"22 99+0"5 98 + 0'87

97 __ 1-22 99+1"22 96 + 1"96

100 __+0'87 93+1-22 100 + 0"05

99 + 1 99+11 94 __ 2"6

100 __ 0 93+6"52 94 + 2-6

100 _____0 100+0 100 __ 0

100 __ 0 100+0 99 + 1-22

100 __ 0 100__0 99 __ 1"22

t.l

12 17 22

Temp.

Copper concentration (l~glitre- l)

(oc) 75 Salinity (%o) 12 17 22

100

14"6

21"9

29"2

14.6

21.9

29"2

100 _ 5'0 99 _ 1"0 100 ___0

97 _ 3"33 99 _ 0"5 98 + 1.94

100 ___0"5 100 + 0-5 100 _ 0

95 _+ 2"55 85 + 4.97 52 + 9"37

27 _ 2.24 25 ___2"87 42 + 9.12

55 -t- 16"57 84 _ 4.74 70 + 5"70

.~ .~

ANOVA

Temp. Linear (TL) Temp. Quadratic (TQ) Salinity Linear (SL) Salinity Quadratic (SQ) Interactions TL x LS TL x SQ TQ x LS TQ x SQ

MS

F-ratio

MS

F-ratio

MS

F-ratio

6-0 10-89 1'5 1.39

1.14 2-05 0"28 0.26

10-67 5.56 10"67 0.87

1.537 0"80 1'537 0-20

0"67 0-22 0-17 0.05

6.01 2.01 1"56 0.45

9 12 0 1

2"25 4"08 4"08 17"36

0"25 0"08 0-08 0"03

4" ~" ~" .~ ,¢ o~

Temp. Linear (TL) Temp. Quadratic (TQ) Salinity Linear (SL) Salinity Quadratic (SQ) Interactions TL x LS TL x SQ TQ x LS TQ x SQ

MS

F-ratio

MS

F-ratio

0.17 0-06 6.72 0-17

0.41 0'14 16.19" 0.41

28.7 122'72 3 556.06 88" 17

0-08 0.34 9-78* 0.24

0 0.33 0-33 1.00

,~"

8.41 280.33 33.33 300.44

MS = Mean Square. * = Significant at 95% confidence level (P < 7.71). to

234

P. T. E. Ozoh, N. V. Jones

and cleavage. The effective fertilization and cleavage started from 18.25%o salinity in the pilot study. The low salinities of 12.2 to 3.86%0 were not favourable to cleavage and fertilization, and 22.88 to 30.5%0 although favourable to fertilization was unfavourable to cleavage (Table 3). The data shown in Table 4 indicate that intermediate and high salinities (14.6 to 29.2%0) favour fertilization and cleavage (Table 5). The mixing of eggs and sperm from several individuals probably increased the chances of fertilization and cleavage. Each experimental salinity contained about 10000 eggs and the sperm which circulate round fertilized eggs probably have better chances of fertilizing unfertilized eggs (see fertilization indices). In Table 4, over 90% fertilization success occurred from 14.6 to 29.2%0 salinity. The ranges of temperature (12 to 22°C) did not affect fertilization success. Temperature and salinity had no significant effect on fertilization at the 95% confidence level (P < 7.71). The 5 and 10 ~g litre- 1 copper had no effect either on fertilization which ranged from 90 to 100%. However, subsequent cleavage stage was below 3%. The copper treatments 0 to 75/~g litre- ~ had no effect on fertilization. The copper level of 100/~g litre-1 significantly influenced the quadratic effect of salinity at the 95% confidence level (P < 7.71). Effect of salinity, temperature and copper on cleavage The data shown in Table 5 indicate the % cleavage success. The intermediate salinity of 14.6%o was favourable to cleavage and high salinity of 29-2%0 was unfavourable when combined with increasing temperature of 22°C. The increasing temperature (12-22°C) with increasing salinity were not favourable to cleavage. At 12°C cleavage success reduced from 88 to 72% with increasing salinities and from 93 to 15% at 22°C in 29.2%0. The ANOVA showed that the linear salinity significantly affected cleavage at the 95% C1 (P<7.71). Low levels of copper (5 to 75/~glitre -1) inhibited cleavage. Better cleavage occurred at 100 #g litre- 1 copper where 10 to 2% success was realized. At 100#glitre -1 copper the linear temperature (LT) and the quadratic salinity (QS) significantly affected cleavage at the 95 % C L (P < 7.71). Many eggs were dead under high temperature and high salinity, with disorganized cell contents and centred cell debris. Effect of salinity, temperature and copper on hatching The data shown in Table 6 indicate hatching success. The hatching in H e d i s t e diversicolor occurred in all the experimental salinities but the best hatching of 93% occurred in 21.9%0 salinity at 12°C. The low temperature of

TABLE 5

Hediste (Nereis) diversicolor Cleavage under Salinity, Temperature and Cu egimes Temp.

Copper concentration (l~glitre- t)

~.

(°c) (x 0

0

100

Salinity (960) (x2) 12 17 22

14.6

21.9

29"2

14"6

21"9

29"2

~"

88 __+3'43 93 + 2"55 86 + 6"7

81 + 4'36 70 + 12"21 67 _+ 13"39

72 _+ 5"79 62 _ 6"42 15 +__2.24

7 ___1"87 3 _ 1.44 2 _ 7'5

10 + 3"64 8 _ 1-44 7 _ 0'87

6 +__3"66 4 ___2'35 5 + 1"5

.~

t~

o

ANOVA

MS LT QT LS QS Interactions LT x LS LT x QS O T x LS O T x QS

888"17 93"39 2 320"67 22"22 756.25 80.08 52"08 117"36

F-ratio

MS

F-ratio

3"53 0"374 9"23* 0"09

13"5

12"16" 2-45 1"35 26'48*

2"72 1"5 29"39 4 0 0 0"44

~" _~"

,~

* = Significant at 95% confidence level (P < 7.71). t.o L~

t~

TABLE 6

Hediste (Nereis) diversicolor Hatching under Salinity Temperature and Copper Regimes Temp. (°C)

Cu concentration (#g litre-1) 0 Salinity (%o) (x 2)

12 17 22

100

14.6

21.9

29.2

14.6

21"9

29.2

73 + 4-74 13 + 6-61 1 +0"35

93 + 1"32 16 + 6-82 0-5+0

68 + 6"61 12 + 1 0+0

4 + 1.8 8 + 1.22 2 + 1.37

7 + 1"87 3 + 0-71 0+0

14 + 2'06 0+0 0+0

.~ .~ .~

ANOVA

MS LT QT LS QS Interaction LT × LS LT × QS TO × LS TQ × QS * Significant at 95% CL (P < 7"71). ** Highly significant at 99'5% (P < 31).

9009"38 1 309"01 8"17 150-22 4 168.75 1-33 26-69

F-ratio 179"51"* 26"08** 0"16 2'99

MS

F-ratio

88"17 0-05 0-00 3"56

4'18 0'002

36 0-33 48 0-11

0'17

"~

Capacity adaptation o f H. d i v e r s i c o l o r to salinity, temperature, copper

Summary

of Optimal

TABLE 7 % Success in the Embryogenesis Salinity Temperature

Developmental state

Optimal % success

of

Hediste ( N e r e i s ) diversicolor u n d e r

and Copper

Optimal temp, (°C)

Regimes

Optimal salinity (%0)

Experimental condition

Fertilization 1.

97-73

16"3

25"98

2.

97-26

24-5

25'56

3.

99'78

7.38

18'3

50 p g l i t r e - 1 C u

4. 5.

98"75 51

9.92 16.9

21.1 23.19

75 p g l i t r e - ~ C u 100/~g litre- t Cu

control 25 p g l i t r e - ~ C u

Cleavage 6.

82"95

10.2

18"92

control

7.

5-2

22.57

25-7

100 # g l i t r e - 1

8.

26-92

28-3

23"52

control

9.

3'9

Hatching

Regression and canonical equations

19"08 employed

8"73

100/~glitre- 1 Cu

to calculate optimal conditions

Fertilization 1.

Y = 9 7 ' 7 3 x x + 0 " 7 8 x 2 - 0"5x 2 - 0 " 2 8 x 2 + l ' 5 X l X

2

Y - Ys = ( ) . 2 6 x 2 - 1.32x~ 2.

Y = 9 7 . 2 6 - 1 . 3 3 x 1 - 1 . 3 3 x 2 + 0-56x~ + 0-22x22 - 0 . 7 5 x l x 2

Y - Ys = 0 . 8 0 2 x 2 + 0 . 0 2 2 x 2 3.

Y = 98.81 - 0 . 3 3 x ~ - O.17x 2 - 0.1 l x 2 + O-06x 2 - 0 . 2 5 x l x 2

Y - - Ys = - 0 " 1 7 6 x 2 + 0 " 1 2 6 x 2 4.

Y = 9 8 ' 7 5 - O ' 1 7 x 1 + 0 . 8 7 x 2 - O.06x 2 + 0"61x2

5.

Y = 51.53 - 2 " 1 7 x l - 3 " 8 3 x 2 - 2 ' 6 7 x 2 +

14"06x2214"5xlx2

Y - Ys = 5 . 3 7 5 x 2 + 16.765x22 Cleavage 6.

Y = 82-95 - 12"17xx - 19-67x2 - 2 - 2 8 x 2 - 1.1 l x 2 - 1 3 . 7 5 X l X 2

Y - Ys = - 8 2 " 5 9 5 x 2 - 5 " 2 1 5 x 2 7.

Y = 5-2 - 1 . 5 x 1 + 0 - 5 x 2 0 . 3 9 x 2 - 1 . 2 8 x 2 +

xlx 2

Hatching 8.

Y = 26"92 - 38"75xa - 1"17x2 + 8 " 5 3 x 2 - 2 " 8 9 x 2 +

Y - Ys = - 8 " 5 5 2 x 2 - 2 " 9 1 x 2 9.

Y = 3"9 - 3 " 8 5 x l - O ' 0 5 x 2

Y - Ys = "1"602x 2 + 1 " 5 4 2 x 2

+ 0"44x 2 - 3xlx2

237

xlx z

238

P. T. E. Ozoh, N. V. Jones

12°C favoured hatching but increasing temperature (12 to 22°C) was not favourable. The hatching success reduced from 93% in 21.9%o salinity at 12°C to 0% in 29.2%0 salinity at 22°C. All the levels of temperature (LT and QT) significantly affected hatching at 95% level from the ANOVA. The 100 #g litre-1 copper depressed hatching. The larvae that hatched out were non-motile or weakly motile and their cilia beat with much reduced rhythmicity. Some larvae seemed to lack cilia and were static. The added copper not only reduced organogenesis but also the movement from monotrochophore to metatrochophore (early and late larva) stages.

Summary of the optimal % success in the embryogenesis The data summarized in Table 7 indicate the % success under the optimal conditions of salinity, temperature and copper. The multiple regression equations and their canonical reductions used in deriving these optima are included. The optimal fertilization success of 97.7 occurred at 16.3°C and 25-98%o salinity, while 90% success level occurred from 0 to 28.2°C and from 8.14 to 38%o salinity. The fertilization in Hediste diversicolor was capacity adapted to low and high temperatures, and low and high salinities. However, with the addition of 25 pg litre- x copper the optimal fertilization success of 97.26% occurred only at 24-5°C and 25-90%o. The 90% success level occurred at 17.6°C and 32%o salinity. The capacity adaptation to temperature and salinity for fertilization was lost with exposure to 25#glitre-1 copper. The adaptation remaining was with respect to high salinity, with 32%o. Equally for 50/~glitre -1 copper the optimal fertilization of 99-78% occurred at 18.2°/oo salinity and 7.38°C. The capacity adaptation to low temperature was retained while that to high salinity was lost. For the 100#glitre-1 copper, the 51% fertilization success occurred from 19.6 to 23.19%o while 50% fertilization occurred from 19-6 to 24"12%o salinity and 16.6 to 18.4°C. These narrow ranges show that capacity adaptations to both temperature and salinity had been destroyed. The cleavage stage was not capacity adapted to high salinity and high temperature. The adaptation was to both the intermediate temperature and intermediate salinity. The added 100/~g litre- ~ copper reduced the cleavage success to only about 5.2% at 22.57%0 and 25.7°C. In hatching success, a combination of high temperature and high salinity was deleterious to hatching since the 26.92% optimal success occurred at 28.2°C and 23.52%o salinity. The hatching success was not capacity adapted to high salinity and high temperature. The 100/~glitre-a copper gave 3.9% hatching success occurring at 10-08°C and 8.73%o salinity.

Capacity adaptation of H. diversicolor to salinity, temperature, copper

239

DISCUSSION The present work was part of a study to determine the effects of copper on the various life cycle stages of Hediste diversicolor under different environmental conditions with a view to consider the potential of this worm as an indicator or biomonitor organism for use in the management of estuaries (Ozoh, 1986). The different stages in the fertilization and embryogenesis were affected differently by salinity, temperature and copper regimes (Table 7. This observation was in conformity with other studies (Tables 1 and 2). Salinity was a 'bottleneck' in the development of Hediste diversicolor (Smith, 1964). In the Scandinavian estuaries, 5 to 8%o favour ripening of the gametes and below 3-4%o Hediste diversicolor cannot reproduce (Bogucki, 1954, 1963). Fertilization occurred at a wide range of salinities (Smith, 1964). This was in conformity with this study, where fertilization was capacity adapted to wide ranging salinities (8.14 to 38%o) and temperatures (0 to 28°C). The cleavage stage occurred from 8 to 27%o (Smith, 1964). This study shows that cleavage was not capacity adapted generally to low and high salinities. The larvae could exist from 8 to 33"5%o salinity (Smith, 1964). However, from this study, they are not capacity adapted to high salinity and high temperature which will limit their abundance. They are adapted to intermediate salinity and adaptation to high temperature, high salinity will probably come with age and maturation since the adults collected from Grimsby exist from 19 to 26%o (Table 1). In Scandinavian estuaries where salinity falls to 4%o, it was very difficult to find worms from the sediments without much searching (Smith, 1964). The added copper concentrations (0 to 100/~glitre -1) destroyed the capacity adaptation shown to the environmental factors of temperature and salinity in the developmental stages, from this study. The use of indicator organisms for environmental quality monitoring depends largely on the ability of the test species to integrate the sediment and the overlying water characteristics. The adult Hediste is an integrator of copper and other heavy metals in the sediment (Bryan, 1971, Bryan et al., 1985). This study has demonstrated that the sensitivity of fertilization and early embryogenesis of Hediste could integrate the overlying water quality. The capacity adaptation of this study confirms the results of other studies of the wide zoogeographical nature of the worm. It is generally agreed that the gametes, embryos and larvae represent the most critical stages in the life cycle of organisms. It1 estuarine water Hediste diversicolor eggs can be fertilized below 8%o salinity but delayed cleavage occurred at this low salinity. The delay might be partly due to the hyperosmoticity of the eggs which took in water osmotically which reduced

240

P. T. E. Ozoh, N. V. Jones

cleavage. However, from 14.6 to 29-2%0 salinity the fertilization proceeded with over 97% success. In the Scandinavian estuaries the gametes ripen at 'critical salinity' in the range of 5 to 8%0 below which animals of marine affinity are not able to survive, grow and reproduce without rather special evolutionary strategy of viviparity (Smith, 1974). Hediste diversicolor cannot reproduce in salinities below 3.4%0 because its larvae cannot develop at low salinities (Bogucki, 1954, 1963; Smith, 1964); the egg does not develop, either. Salinity may modify the time and length of breeding seasons and the rates of embryonic development (Broekhuysen, 1936). The fertilization in Hediste diversicolor was not affected by copper concentrations from 0 to 75/~g litre- 1 but 100 #g litre- 1 copper destroyed the capacity adaptation to salinity and temperature (Table 7). This inability to respond to changing salinity and temperature common in estuaries will have a limiting effect on the zoogeography of the worm. The literature survey does not reveal any study on the effects of pollutants in general and copper in particular on the developmental stages of Hediste diversicolor. However, copper can be very toxic to the developmental stages of a number of species. Copper concentration of 10 #g litre- ~ induced 0.9% bifurcated abnormal larvae in Capitella capitata and reduced the numbers of viable females and larvae at increasing exposure levels (Reish et al., 1974). The suppression of reproduction was noted at 0-5 mg litre-~ Cu in Ctenodrilus serratus and Ophryotrocha diadema (Reish & Carr, 1978). The adults and juveniles of Neanthes arenaceodentata were equally affected by copper at the 96h exposure level but at 28 days the juveniles were more sensitive than the adults. The trochophore larvae of Capitella capitata were more sensitive to copper than the adults (Reish et al., 1976). Copper inhibited fertilization and showed differential reaction to the ova and sperm in sea urchin (Lee & Xu, 1984). Copper doses of 0.1 and 0.5 ppm blocked fertilization, the sperm were more sensitive than the ova and 0.0031 ppm Cu induced abnormal development (Lee & Xu, 1984). Murakami et al. (1976) reported complete inhibition at 10 -5 mole copper. Ten micrograms per litre copper reduced fecundity and the viability of herring eggs (Steel et al., 1973) and 100/2g litre- ~copper gave 50% mortality in 48 h to Crassostrea virg&ica (Calabrese et aL, 1973). Capitella capitata trochophore suffered 50% mortality in 96 h at 180 #g litre- ~ copper solution (Reish et al., 1976). Copper at 3-4/~g litre- 1 inhibited development in zebra fish eggs and caused spirality of the notochord and other abnormalities (Ozoh, 1979). The debilitating effects of copper on development were not limited simply to embryonic and larval mortality as the defective larvae generally increased with high exposure concentrations (Birge & Black, 1976). In the coral reef echinoid, Echinometra mathaei, 50% reduction of fertilization success and cleavage to the eight-cell stage occurred at 0"18 and

Capacity adaptation of H. diversicolor to salinity, temperature, copper

241

0.42 mg litre- 1 added copper, respectively. The larval skeletal development was suppressed at 0.02mglitre -1 even though copper was sub-lethal because the larval mortality was under 10% (Heslinga, 1971). In the bivalve, C. crassastrea 10 #g litre-1 Cu affected development ,and 50#glitre -1 Cu solution reduced fertilization success by 25% of the control in the echinoid. At 220#g litre -1 fertilization success was reduced by 58% and only about 0.05 % developed fertilization membranes at the highest added copper concentration of 670/~glitre -1 (Okubu & Okubu, 1962). In the Chlorella pyrenoidosa copper inhibited cleavage and if the cells initially dividing were exposed to copper they continued division but if the initial division had not started, subsequent division would not occur (Steeman-Nielsen, et al., 1969). Waterman (1937) found fertilization to be more sensitive than cleavage in the eggs of temperate echinoid and marked inhibition of fertilization started at 190#glitre -1, while 760#glitre -1 copper suppressed cleavage. Kobayashi (1971) reported both fertilization and cleavage in the temperate echinoid, Anthrocidaris crassispina to be retarded at copper concentrations between 100 and 200/~g litre- 1. Besides copper, low salinity can inhibit fertilization and cleavage in some marine invertebrates. Tait et al. (1984) reported that at 60% sea water meiosis could not occur in the polychaete, Galeolaria caespitosa, but normal development proceeded in normal or 80% sea water. Pollution at sub-lethal levels may not affect the adult, but can affect the gametes, embryos and larvae since those represent the most sensitive stages of the life cycle. That copper and salinity can affect differently these stages highlights the complexity in setting water quality parameters in pollution monitoring.

A C K N O W L E D G E M ENTS The grant came from the Association of Commonwealth Universities as Academic Staff Scholarship to one of us (P.T.E.O). Chris Park collected the specimens.

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